Generation and use of dendritic cells

ABSTRACT

The present invention relates to an in vitro method for differentiation and maturation of isolated monocytes into IL3-R+CD11c+ myeloid dendritic cells consisting of incubating the monocytes with a combination of a type I interferon and IL-3.

This application is a 35 U.S.C. 371 National Stage application ofPCT/EP01/13189, published in English under PCT Article 21(2), andclaiming the benefit of European Application 00870273.0, filed Nov. 14,2000. The above applications are incorporated by reference in theirentirety.

TECHNICAL FIELD

The present invention relates to the improvement of cell therapy for thetreatment or prevention of cancer, infections and autoimmune diseases inparticular in the development of new dendritic cells carrying superiorcharacter in eliminating or preventing the occurrence of invasive cells.

BACKGROUND ART

Dendritic cells (DC) are key players in the initiation of immuneresponses. This cell type constitutes the most potent antigen-presentingcell, endowed with the unique capacity to cluster naïve T cells.Consequently, it has been proposed as a natural adjuvant, aiming at thetriggering of T-cell responses against poor immunogens, such astumor-associated Ag (TAA). The realization of clinical trials has longbeen impaired by the low frequency of circulating DC available in theblood. The development of methods of generating large numbers of DC fromhemapoietic precursors has recently allowed the initiation of pioneerclinical trials. These trials have yielded promising results for cancertherapy.

Dendritic cells (DC) represent a major class of antigen-presenting cellscharacterized by their unique ability to prime naive T cells¹. Recentworks demonstrated the existence of several DC subsets whichdifferentiate from either lymphoid or myeloid bone marrowprogenitors^(2;3). A critical factor for myeloid DC development isgranulocyte-macrophage colony-stimulating-factor (GM-CSF)^(4;5) whereaslymphoid DC are dependent on interleukin (IL)-3 for theirsurvival^(6;7). On the basis of the expression of myeloid markers (i.e.CD11c) or IL-3 receptor-α (IL-3Rα) chain (CD123) expression, two typesof DC precursors have been isolated from human peripheral blood⁷. Onesubtype displays myeloid surface markers and low levels of IL-3Rα,whereas another subtype of putative lymphoid origin express IL-3Rα, isexquisitely dependent on IL-3 for its survival and is a strong producerof type I interferons (IFNs).

Human myeloid DC can be easily generated in vitro by culturing monocytesin presence of GM-CSF and IL-4^(4;5) whereas the so-called lymphoid DChave been obtained by isolation of precursors from blood or lymphnodes^(6;7).

The GM-CSF/IL4 DC have been used in clinical trials in cell therapy⁴⁴.However, using these conditions, only 67% of immunized patients showedan increase in response to the treatment. Therefore it is important toimprove the method, conditions or substances to improve the efficacityof the treatment.

SUMMARY OF THE INVENTION

The present invention is directed towards providing a method for theproduction of superiour dendritic cells which can be used in celltherapy to eliminate or prevent more efficiently the deleterious effectsof invasive cells in patients. The inventors found surprisingly thatwhen monocytes were incubated in specific conditions in presence of IL3and IFN-β a superior type of DC could be produced.

Type I IFNs are produced by several cell types in response to viral,bacterial and protozoan infections⁸⁻¹³. Through their multiple effectson natural killer cells and T cells, type I IFNs represent a criticallink between innate and acquired immunity¹⁴. Recent studies indicatethat type I IFNs might also influence DC differentiation andmaturation^(15;16). Indeed, IFN-β was shown to promote monocytedifferentiation into short-lived DC rapidly undergoing apoptosis¹⁶.Present inventors investigated whether apoptosis could be stopped usingIL-3. To study this, the inventors determined the effect of IFN-β on theexpression of IL-3Rα on monocytes and characterized the phenotype and Tcell stimulatory capacity of cells derived from monocytes cultured inpresence of IL-3 and IFN-β. The present inventors found surprisinglythat indeed IL-3 could rescue IFN-β treated cells. In addition thesecells seemed to be superior in inducing the immune system.

A particular object of the present invention is directed towards a newtype of stable dendritic cell (DC) which is more mature and is morepotent in activating the immune system compared to other known stableDCs. Monocytes cultured in IL3 and IFN-β differentiate into dendriticcells with potent T cell stimulatory activities. This invention derivesfrom the original and unexpected finding that IFN-β maintains IL3Rexpression on monocytes. IL3 allows survival of DC which are short-livedwhen generated in IFN-β alone and are therefore not suitable for celltherapy. These cells induce both TH1-type (IFN-γ) and TH2-type responses(IL-5), which might be especially advantageous in cancer immunotherapy.These cells directly induce apoptosis of certain tumor cells. Thepresent inventors propose to use those cells for cancer immunotherapyand vaccination against infectious pathogens either by loading the DCsex vivo with tumor proteins, peptides, gene-transfer (RNA, DNA), fusionwith tumor cells (hybrids), followed by the in vivo injection, or toinject the cell directly in the tumor where they could induce therelease of tumor antigens through their killing activity.

These aims have been met by following embodiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for differentiation andmaturation of monocytes into IL3-R+ CD11c+ myeloid dendritic cellscomprising incubating said monocytes with a combination of IFN-β andIL-3 (or IL3), or functional analogues thereof. With functionalanalogues it is meant IFN-α or other type I IFNs as known by a skilledperson in the art; also peptidomimetic molecules might be used for thispurpose. The inventors proved that IFN-α exerts a similar effect asIFN-β, as the IL-3+IFN-α combination also resulted in the generation ofa comparable myeloid IL-3Rα-positive DC (FIG. 5). When monocytes werecultured in IL-3 alone, the inventors found that they expressed lowerlevels of CD80 and CD86 and higher levels of CD14 than (L-3/IFN-β DC(FIG. 4), which suggests that they are more close to macrophages.

The IL-3/IFN-β DC (or IFN-β/IL-3 DC) cell type resulting from saidmethod is new and differs significantly from previous obtained stableIL4/GM-CSF DC. Monocytes purified from PBMC (peripheral bloodmononuclear cells), which are the precursor cells of DC, express IL-3Rα(CD123) but lose this expression after culture in medium alone. Presentresults shows that the loss of IL-3Rα expression was prevented whenIFN-β was added during the culture. Nevertheless, more than 90% ofmonocytes cultured in medium alone or in presence of IFN-β were found toundergo apoptosis. The inventors found surprisingly that the addition ofIL-3 in the presence of IFN-β dramatically enhanced cell survival.Indeed, more than 65% of cells were still alive using these culturingconditions. Present inventors found surprisingly that IL-3 rescuemonocytes cultured in the presence of IFN-β from apoptosis, and allowsthem to further differentiate. These observations demonstrate acooperating effect of IL-3 and IFN-β on cell survival anddifferentiation. The effect of type I IFNs on IL-3Rα expression hasnever been assessed before.

To characterize the cells obtained by culture of monocytes in IL-3 andIFN-β, the present inventors first looked at their ultrastructuralmorphology and found that monocytes cultured under the conditionsacquired cytoplasmic expansions of the dendritic type. Cells derivedfrom monocytes cultured in IL-3 and IFN-β can therefore be referred tohereafter as IL-3/IFN-β DC or IFN-β/IL-3 DC.

The present inventors further characterized that IL-3/IFN-β DC expressedmarkers of the myeloid lineage (CD11c, CD14, and CD33). They alsoexpressed high levels of HLA class I and class II molecules, CD40, CD54,CD 80 and CD86, and IL-3Rα (CD123). Contrary to IL-4/GM-CSF DC,IL-3/IFN-β DC showed much higher levels of IL-3Rα. Conversely, CD1a wasexpressed on IL4/GM-CSF DC but not on cells derived from monocytescultured in IL-3 and IFN-β. The present results therefore indicated thatIL-3/IFN-βDC are also myeloid but phenotypically completely differentcells compared to the known IL4/GM-CSF DC. Based on their markersIL-3/IFN-β DCs may also be referred as IL3-R+ CD11c+ dendritic cells(DC) or IL3-R+ CD11c+ myeloid dendritic cells.

Advantageously, IL-3 and IFN-β can be added to the monocytessimultaneously, sequentially or separately.

According to the invention IFN-β is present at a concentration ofbetween 10 and 20000 U/ml.

The present results demonstrates that the loss of IL-3Rα expression wasprevented by adding 1000 U/ml IFN-β in the culture medium. Nevertheless,concentrations of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95,100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1150, 1200, 1250,1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850,1900, 1950, 2000, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500,2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3100, 3150,3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750,3800, 3850, 3900, 3950, 4000, 4100, 4150, 4200, 4250, 4300, 4350, 4400,4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950, 5000,5100, 5150, 5200, 5250, 5300, 5350, 5400, 5450, 5500, 5550, 5600, 5650,5700, 5750, 5800, 5850, 5900, 5950, 6000, 6100, 6150, 6200, 6250, 6300,6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900,6950, 7000, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550,7600, 7650, 7700, 7750, 7800, 7850, 7900, 7950, 8000, 8100, 8150, 8200,8250, 8300, 8350, 8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800,8850, 8900, 8950, 9000, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450,9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950, 10000,10050, 10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500,10550, 10600, 10650, 10700, 10750, 10800, 10850, 10900, 10950, 11000,11050, 11100, 11150, 11200, 11250, 11300, 11350, 11400, 11450, 11500,11550, 11600, 11650, 11700, 11750, 11800, 11850, 11900, 11950, 12000,12050, 12100, 12150, 12200, 12250,.12300, 12350, 12400, 12450, 12500,12550, 12600, 12650, 12700, 12750, 12800, 12850, 12900, 12950, 13000,13050, 13100, 13150, 13200, 13250, 13300, 13350, 13400, 13450, 13500,13550, 13600, 13650, 13700, 13750, 13800, 13850, 13900, 13950, 14000,14100, 14150, 14200, 14250, 14300, 14350, 14400, 14450, 14500, 14550,14600, 14650, 14700, 14750, 14800, 14850, 14900, 14950, 15000, 15050,15100, 15150, 16200, 15250, 15300, 15350, 15400, 15450, 15500, 15550,15600, 15650, 15700, 15750, 15800, 15850, 15900, 15950, 16000, 16050,16100, 16150, 16200, 16250, 16300, 16350, 16400, 16450, 16500, 16550,16600, 16650, 16700, 16750, 16800, 16850, 16900, 16950, 17000, 17100,17150, 17200, 17250, 17300, 17350, 17400, 17450, 17500, 17550, 17600,17650, 17700, 17750, 17800, 17850, 17900, 17950, 18000, 18050, 18100,18150, 18200, 18250, 18300, 18350, 18400, 18450, 18500, 18550, 18600,18650, 18700, 18750, 18800, 18850, 18900, 18950, 19000, 19050, 19100,19150, 19200, 19250, 19300, 19350, 19400, 19450, 19500, 19550, 19600,19650, 19700, 19750, 19800, 19850, 19900, 19950 and 20000 U/ml arepossible.

According to a preferred embodiment of the invention IFN-β is given at aconcentration of 1000 U/ml.

According to the invention IL-3 is present at a concentration between 1and 1000 U/ml. In this respect, concentrations of 1, 2, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315,320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385,390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455,460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525,530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595,600, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765,770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835,840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905,910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975,980, 985, 990, 995 and 1000 U/ml are possible.

According to a preferred embodiment of the invention, IL-3 is present ata concentration of 50 U/ml.

According to the present invention the method to provide a population ofIL3-R+ CD11c+ myeloid dendritic cells comprises at least the followingsteps:

-   (a) isolating monocytes from a patient, and,-   (b) incubating said monocytes in the presence of IFN-β and IL-3 or    functional analogues thereof.

There are two main sources of DC precursors: CD34+ stem cells andperipheral blood (PB) monocytes. The main constraints of generating DCfrom stem cells is that the culture time is long and obtaining CD34+cells requires mobilization of the patient. Therefore a preferredembodiment of the present invention is to use monocytes as a DCprecursor. These cells can normally, when present in blood,differentiate into DC in presence of GM-CSF or as the present inventorsshowed in presence of IL3-IFN-β. Different techniques might be used toisolate monocytes from the blood as known by the person skilled in theart. A preferred method is described in example 1. With the term“population” is meant IL3-R+ CD11c+ myeloid dendritic cells as such, agroup of IL3-R+ CD11c+ myeloid dendritic cells which may be different inother characteristics, or a group of cells comprising IL3-R+ CD11c+myeloid dendritic cells. Also one cell is not excluded from thisdefinition. It is important to mention that, the inventors do notexclude the fact that monocytes can be further differentiated andmaturated in vivo by injecting IL-3 and IFN-β as such into the patient.

According to present invention IL3-R+ CD11c+ myeloid dendritic cells mayfurther be treated to produce antigen presenting dendritic cells.Consequently the method as described by the present invention comprisesat least the following steps:

-   (a) isolating monocytes from a patient,-   (b) incubating said monocytes in the presence of IFN-β and IL-3    producing IL3-R+ CD11c+ myeloid dendritic cells, and,-   (c) presenting an antigen on the surface of said dendritic cells.

Depending on the specific treatment as described below, antigens arespecific molecules present on cells selected from the group consistingof a cancer cell, a bacteria, a parasitically infected cell and avirally infected cell. These antigens can be large molecules which areprocessed by the DC to load MHC molecules, or can be smaller molecules(i.e. peptides) which are immediately loaded onto the MHC molecules.Several approaches have been used to arm DC with target antigen for usein clinical trials. Methods used to approach this step of antigenloading are reviewed by Fong and Engleman⁴⁴. Inventors also point outthat, IL3-R+ CD11c+ myeloid dendritic cells can be produced in vivo byinjecting IL-3 and IFN-β or IL-3 and IFN-β in combination with anantigen into the patient. The capacity of presenting a peptide on thesurface of said dendritic cells according to present invention can forexample be achieved by contacting said dendritic cell with at least partof an antigen differentially expressed on a cell. This cell can be acell selected from the group consisting of a cancer cell, a bacterialcell, a parasitically infected cell and a virally infected cell.Antigens are delivered from these to the DC resulting in the activationof the DCs.

Alternatively, the capacity of presenting a peptide on the surface ofsaid dendritic cells can be achieved by pulsing said dendritic cellswith antigenic proteins, by loading said dendritic cells with antigenicpeptides or can be achieved by transforming/transducing said dendriticcells by nucleic acid molecules coding for at least part of saidantigen. With “pulsing” is meant that DC are activated by these antigensand enter into the MHC class II and/or MHC class I processing pathway.Transformation of DC can be achieved using electric pulses, liposomes orother techniques as known by the person skilled in the art. Viralvectors allow the transduction of cells. With viral vectors alsoretroviral, adenoviral and adeno-associated vectors are meant.

Transformation/transduction of the cells allows introduction of DNAencoding the antigen and when appropriate expression signals are presentsaid antigen is made in the cell and brought through the endogenousmechanisms to the surface of the transformed/transduced dendritic cell.As a result of this an antigen-presenting IL3-R+ CD11c+ myeloiddendritic cells is made.

Alternatively, the capacity of presenting a peptide on the surface ofsaid dendritic cells is achieved by fusing said dendritic cell withcells carrying specific antigens. The production of dendritic-likecell/tumor cell hybrids and hybridomas for inducing anti-tumor responsehave been described in WO96/30030. This document provides dendritic-likecell/tumor cell hybridomas and pluralities of dendritic-like cell/tumorcell hybrids that confer tumor resistance in vivo. The hybrids andhybridomas are generated by the fusion of tumor cells withdendritic-like cells. For instance, immortal tumor cells from anautologous tumor cell line can be fused with autologous or HLA-matchedallogeneic dendritic-like cells. Autologous tumor cell lines can bederived from primary tumors and from their metastases. Alternatively,immortal dendritc-like cells from an autologous or allogeneicHLA-matched dendritic-like cell line can be fused with autologous tumorcells. Autologous dendritic-like cell lines can be prepared from varioussources such as peripheral blood and bone marrow. Dendritic-likecell/tumor cell hybridomas and pluralities of hybrids can be directlyinfused for active immunization of cancer patients against theirresidual tumor cells. The hybridomas and hybrids can also be used forthe in vitro activation of autologous immune cells before theirreinfusion into the patient for passive immunization against the tumorcells.

The present invention also proposes a method to provide an activatedpopulation of T cells using antigen-presenting IL3-R+ CD11c+ myeloiddendritic cells obtainable by a method as described above comprising atleast the following steps:

-   (a) isolating monocytes from a patient,-   (b) incubating said monocytes in the presence of IFN-β and IL-3 to    provide a population of IL3-R+ CD11c+ myeloid dendritic cells,-   (c) presenting an antigen (for instance peptide or protein) on the    surface of said dendritic cells, thereby providing a population of    antigen presenting dendritic cells; and,-   (d) activating a population of T cells with said population of    antigen presenting dendritic cells.

An activated T cell being a T cell (CD3+ cell) proliferating and/orsecreting cytokines (IL-2, IL4, IL-5, IFN-γ, etc.) and/or expressingactivation markers (CD25, CD69, HLA-DR, CD40L, etc.). Indeed, thepresent inventors indicated that the low levels of IL-12 secreted byIL-3/IFN-β DC contribute to their ability to elicit IFN-γ production byT cells. In addition, the inventors proved that, IL-3/IFN-β DC alsoinduced IL-5 production in mixed leucocyte culture and were also muchmore efficient than IL-4/GM-CSF DC in that respect. If necessary, anactivated T cell can always be separated from the antigen presentingdendritic cell by cell sorting.

In preferred methods according to present invention said T cell is a Thelper cell.

The invention also refers to a method wherein the steps of producing apopulation of cells as described above such as IL3-R+ CD11c+ myeloiddendritic cells and/or antigen presenting IL3-R+ CD11c+ myeloiddendritic cells and/or activated T cells using antigen presenting IL3-R+CD11c+ myeloid dendritic cells occur in vitro and/or in vivo.

The present invention also provides a population of IL3-R+ CD11c+myeloid dendritic cells, a population of antigen-presenting IL3-R+CD11c+ myeloid dendritic cells or a population of activated T cellsobtainable by a method as described above, or a combination thereof.

The present inventors showed that, although IL-3/IFN-β DC displayseveral features suggestive of a higher degree of maturation, theyshould not be considered as fully mature. Indeed, the inventors gaveevidence that the IL3-R+ CD11c+ myeloid dendritic cells according topresent invention can be further stimulated using a stimulant. Thepresent invention also refers to a method to further stimulate apopulation of IL3-R+ CD11c+ myeloid dendritic cells, whereby saiddendritic cells are additionally incubated with a stimulant. Thisincubation can be performed simultaneously, sequentially or separatelyfrom the cytokine treatment. With stimulation is also meant maturation,induction and/or activation. According to present invention, saidstimulant may be chosen from a group comprising virus, bacterium, LPS(lipopolysaccharide), nucleic acid, functional derivatives or acombination thereof. The term “nucleic acids” refers to a singlestranded or double stranded nucleic acid sequence, said nucleic acid mayconsist of deoxyribonucleotides (DNA) or ribonucleotides (RNA) or may beamplified cDNA or amplified genomic DNA. Consequently, the presentinvention also relates to a population of stimulated IL3-R+ CD11c+myeloid dendritic cells obtainable by a method according to presentinvention. In the examples, the inventors showed that when analyzing theIFN-α production by IL-3/IFN-β DC, stimuli such as LPS andformaldehyde-inactivated influenza virus induced said DC only to a lowerextent (table 2). Contrarily, Poly (I:C), which mimicks viraldouble-stranded RNA, was able to induce IFN-α production by IL-3/IFN-βDC to a higher extent. It has to be understood that all applications(such as methods, compositions, kits, uses) suggested in presentinvention for unstimulated IL-3/IFN-β DC may be applied for stimulatedIL-3/IFN-β DC.

The present invention relates to a composition for use as a medicamentor cell based product intended for clinical use comprising at least oneof the following combinations of components:

-   -   IL-3 and IFN-β or functional analogues thereof,    -   IL-3 and IFN-β or functional analogues thereof mixed with        antigen and/or stimulant,    -   a population of monocytes mixed with IL-3 and IFN-β or        functional analogues thereof,    -   a population of monocytes mixed with IL-3, IFN-β or functional        analogues thereof and antigen and/or stimulant,    -   a population of IL3-R+ CD11c+ myeloid dendritic cells,    -   a population of IL3-R+ CD11c+ myeloid dendritic cells mixed with        antigen and/or stimulant,    -   a population of antigen-presenting IL3-R+ CD11c+ myeloid        dendritc cells, or,    -   a population of activated T cells obtainable using        antigen-presenting IL3-R+ CD11c+ myeloid dendritic cells,        whereby said IL3-R+ CD11c+ myeloid dendritic cells are        unstimulated or stimulated using a method according to present        invention, or a combination thereof.

Cell based products are not yet considered as medicament and could beconsidered as transfusion products in the future.

The present results also showed that the IL-3/IFN-β DC have endocytosiscapacity. IL-3/IFN-β DC spontaneously secreted IL-6, IL-8, IL-12 (p40)and TNFα.

The inventors also studied the cytokine production of GM-CSF/IL-4 DCversus IL-3/IFN-β DC. As compared to GM-CSF/IL-4 DC, IL-3/IFN-β DCproduced less IL-12 (p40) whereas their secretion of IL-8 was slightlyhigher. As it is the case for IL-4/GM-CSF DC, both LPS and CD40 ligationinduced by CD40L transfectants upregulated the synthesis of cytokines byIL-3/IFN-β DC. As compared to GM-CSF/IL-4 DC, IL-3/IFN-β DC producedsimilar levels of TNFα and IL-6 levels under both conditions ofstimulation, higher IL-8 levels in response to CD40L but not to LPS, andmuch lower levels of IL-12 (p40) and IL-12 (p70) whatever the stimulusconsidered.

At all stimulator/responder ratios, IL-3/IFN-β DC were as efficient asGM-CSF/IL-4 DC to induce naive CD4⁺ T cell proliferation. Nevertheless,if the inventors considered the profile of cytokines secreted by T cellsupon exposure to allogenic DC, IL-3/IFN-β DC induced the production ofsubstantial amounts IFN-γ, despite their low synthesis of IL-12. Inaddition, IL-3/IFN-β DC also elicited IL-5 production and weresignificantly more efficient than IL4/GM-CSF DC in that respect.

Despite their low level of IL-12 production, IL-3/IFN-β DC stimulatehigh level of IFN-γ production from adult CD4⁺ T cells, suggesting thatthey use other factors or membrane molecules to elicit the synthesis ofTh1 cytokines. Indeed IL-12 independent pathways of IFN-γ were recentlydescribed^(13;14;28;29). Taken together, these data indicate thatIL-3/IFN-β DC differ from the CD4⁺ CD3⁻ CD11c⁻ IL-3Rα⁺ plasmacytoidcells isolated from peripheral blood described by Grouard et al.⁶, asthe latter cells proved to be poor inducers of IFN-γ in MLR.

The present inventors conclude that upon using above described methodsnew types of DC can be formed which are more mature than previouslydescribed myeloid DC and which have a superior character in inducing Tcell expression. Increased cytokine expression results in a more rapidand efficient stimulation of the immune system, and therefore will bemore efficient in eliminating foreign infectious material in a patient.

Monocyte-derived DC primed with tumor antigens are now clinically usedin several protocols to induce specific antitumor immunity³⁰⁻³². BothTh1 and Th2 effector mechanisms have been shown to collaborate with eachother in directing an effective antitumor activity³³. Because of theirability to induce both Th1 and Th2 type responses, the inventors suggestthat IFN-β/IL-3 DC (induced or uninduced) might be appropriate to induceefficient tumor immunity.

Preferably, a composition according to the invention is supplementedwith at least one additional cytokine. According to the presentinvention, said cytokine is preferentially chosen from a groupcomprising IFN-α, IFN-β, IL-3 and IL-12. IL-12 and IFN-α are pivotalcytokines for Th1 differentiation and generation of cytotoxic T cellsendowed with potent anti-tumor effects.

The invention implies the preparation of a medicament for treatingcancer, infections and autoimmune diseases comprising a composition asdescribed above. Investigations showed that the immunologic and clinicaleffects of antigen-loaded dendritic cells administered as a therapeuticvaccine to patients with cancer⁴⁴. Although DC-based vaccination methodsare cumbersome, promising results from clinical trials in patients withmalignant lymphoma, melanoma, and prostate cancer suggest thatimmunotherapeutic strategies that take advantage of theantigen-presenting properties of dendritic cells may ultimately proveboth efficacious and widely applicable to human tumors. Also the role ofDC in initiating or priming immune responses to viral and bacterialantigens in vivo is well established. It has been demonstrated thathuman DC, but not monocytes or B cells, can sensitise naïve T cells tosoluble protein antigens, enabling the generation of antigen-specificCD4+ helper and CD8+ CTL lines in vitro⁴⁴. CD8+ cytotoxic T lymphocytes(CTL) have been demonstrated to recognize and kill cancer cells invarious tumor models. The ability of DC to prime T cells capable ofrecognizing and killing tumor cells in an antigen-specific fashion hasbeen demonstrated in various animal models. Moreover, DC-basedimmunization can lead to immunologic memory with protection againstsubsequent tumor challenges. Fong et al (1997⁴⁵) illustrated thatimmunizing with self proteins could protect animals against autoimmunereactions.

The present invention also relates to the pharmacological compositioncomprising the composition according to the invention and optionally apharmaceutical acceptable carrier, diluent or excipient.Pharmaceutically acceptable carriers include any carrier that does notitself induce the production of antibodies harmful to the individualreceiving the composition. Suitable carriers are typically large, slowlymetabolizing macromolecules such as proteins, polysaccharides,polylactic acids, polyglycolic acids, polymeric amino acids, amino acidcopolymers; and inactive virus particles. Such carriers are well knownto those of ordinary skill in the art. A “vaccine” is an immunogeniccomposition capable of eliciting protection against infections, whetherpartial or complete. A vaccine may also be useful for treatment of anindividual, in which case it is called a therapeutic vaccine. Saidvaccine compositions may include prophylactic as well as therapeuticvaccine compositions. The term “therapeutic” refers to be capacity ofeliminating or preventing invasive cells.

The present invention also relates to a method of killing a target cellcomprising contacting said target cell with a composition. This killingcan be performed in vitro or in vivo.

Preferably, said target cell is selected from the group consisting of acancer cell, a bacterial cell, a parasitically infected cell or avirally-infected cell.

The present invention also provides an in vitro screening method using apopulation of IL3-R+ CD11c+ myeloid dendritic cells, a population ofantigen-presenting IL3-R+ CD11c+ myeloid dendritic cells, a populationof stimulated IL3-R+ CD11c+ myeloid dendritic cells, a population ofstimulated antigen-presenting IL3-R+ CD11c+ myeloid dendritic cells or apopulation of activated. T cells obtainable by a method as described inpresent invention. By their potent immunostimulatory properties, DCloaded with tumor or bacterial Ag could be used to activate T cellsagainst unknown poorly immunigenic Ag and thus help to discover them.

According to present invention, a population of myeloid IL3-R+ CD11c+dendritic cells, a population of antigen-presenting IL3-R+ CD11c+myeloid dendritic cells, a population of stimulated IL3-R+ CD11c+myeloid dendritic cells, a population of stimulated antigen-presentingIL3-R+ CD11c+ myeloid dendritic cells or a population of activated Tcell using antigen-presenting IL3-R+ CD11c+ myeloid dendritic cellsobtainable by a method according to the present invention can be usedfor the preparation of in vitro screening tests.

According to present invention, a method for detecting T cell mediatedactivity of a target antigenic peptide comprises at least the followingsteps:

-   (a) providing a population of antigen-presenting IL3-R+ CD11c+    myeloid dendritic cells obtainable from monocytes according to a    method as described above,-   (b) contacting a T cell with said dendritic cell thereby providing    an activated T cell,-   (c) contacting a target cell with said activated T cell, and,-   (d) monitoring the effect of said activated T cell on said target    cell, thereby detecting anti-target activity,    whereby said IL3-R+ CD11c+ myeloid dendritic cells are unstimulated    or stimulated using a method according to the present invention, or    a combination thereof.

The present invention also describes a kit for detecting T cell mediatedactivity of a target antigenic peptide, comprising at least onecombination of components chosen from the following list:

-   -   IL-3 and IFN-β, or functional analogues thereof,    -   IL-3 and IFN-β or functional analogues thereof mixed with        antigen and/or stimulant,    -   a population of monocytes mixed with IL-3 and IFN-β or        functional analogues thereof,    -   a population of monocytes mixed with IL-3, IFNβ or functional        analogues thereof and antigen and/or stimulant,    -   a population of IL3-R+ CD11c+ myeloid dendritic cells,    -   a population of IL3-R+ CD11c+ myeloid dendritic cells mixed with        antigen and/or stimulant,    -   a population of antigen-presenting IL3-R+ CD11c+ myeloid        dendritic cells, or    -   a population of activated T cells obtainable using        antigen-presenting IL3-R+ CD11c+ myeloid dendritic cells        whereby said IL3-R+ CD11c+ myeloid dendritic cells are        unstimulated or stimulated using a method according to the        present invention, or a combination thereof.

It has been shown that freezing population of said cells did not alterthe functional properties of these cells. Also other methods for storageas known by the skilled person in the art can be applied to preservethese cells⁴⁶.

All methods, uses and kits described in the present invention for thedetection of T cell mediated activity also relate to the use of apopulation of IL3-IFN-β DC as reagent for the purpose of following theimmune response in patients who got either DC-vaccines or othervaccines. T cells might be isolated from patients and tested usingantigen-presenting IL3-IFN-β DC to analyse if the immunologic responsein the patient has been activated. For his purpose PBMC (periferal bloodmononuclear cells) or purified T cells might be used. This test systemallows the evaluation of any therapy against infections, cancer orauto-immune diseases.

The present invention suggests the use of a composition according to theinvention as a vaccine adjuvant and the vaccine adjuvant as suchcomprising a composition according to the invention.

According to present invention a vaccine comprising the composition asdescribed by the invention can be used to immunize humans or animalsagainst different diseases. Vaccination of patients has already beenillustrated and found to be efficacious using peptide-pulsed IL4/GM-CSFDC in cancer patients (Toungouz et al. 1999⁴⁶).

In particular, the present invention describes a method for immunizinghumans or animals against a disease comprising administering a vaccinecomprising an adjuvant as described above.

The present invention also relates to a method of treatment of cancer,infections and autoimmune diseases comprising the use of at least one ofthe following combinations of components:

-   -   IL-3 and IFN-β or functional analogues thereof,    -   IL-3 and IFN-β or functional analogues thereof mixed with        antigen and/or stimulant,    -   a population of monocytes mixed with IL-3 and IFN-β or        functional analogues thereof,    -   a population of monocytes mixed with IL-3, IFN-β or functional        analogues thereof and antigen and/or stimulant,    -   a population of IL3-R+ CD11c+ myeloid dendritic cells,    -   a population of IL3-R+ CD11c+ myeloid dendritic cells mixed with        antigen and/or stimulant,    -   a population of antigen-presenting IL3-R+ CD11c+ myeloid        dendritic cells, or,    -   a population of activated T cells obtainable using        antigen-presenting IL3-R+ CD11c+ myeloid dendritic cells        whereby said IL3-R+ CD11c+ myeloid dendritic cells are        unstimulated or stimulated using a method according to the        present invention, or a combination thereof.

In the design and conduct of above described applications, importantconsiderations include methods for introducing the antigen into MHCclass I and II processing pathways, methods for isolating and activatingdendritic cells, route of administration and antigen selection. Becausethe cell therapy as presented in the present invention needs a specificrecognition of the target cell, it is important that indeed the choiceof antigen is well considered. Therefore the present invention suggeststhat the antigen is a tumor specific antigen, an infectious specificantigen or a self-protein when applied in the treatment of cancer,infections (viral, bacterial, parasitical) or autoimmune diseases. Inaddition, it is important that the compositions are administered to aperson in need of treatment in a therapeutically effective amount.Example of antigens that might be considered as tumor antigens aredescribed by Fong and Engleman 2000⁴⁴.

According to the present invention said viral disease is selected fromthe group consisting of for instance HIV, human Papilloma virus, EbsteinBarr virus and Cytomegalovirus.

According to the present invention said autoimmune disease is selectedfrom the group consisting of multiple sclerosis myasthenia gravis,juvenile chronic arthritis, chronic arthritis, LED, atopic dermatitisand juvenile diabetes. Inventors suggest that probably all autoimmunediseases may be treated or prevented by a method as described by theinvention.

According to the present invention, said compositions can be injectedinto patients using different ways. Preferentially injection is carriedout intravenously, intra-lymphoidal or intratumoral, nevertheless, otherroutes can be used such as subcutaneous injections. It is interesting tomention that in addition to expressing the requisite MHC andcostimulatory molecules to prime T cells, the DC cells expressappropriate adhesion molecules and chemokine receptors to attract the DCto secondary lymphoid organs for priming. In this respect, inefficientpriming could be circumvented by injecting DC directly to secundarylympoid organs through intralymphatic or intranodal injection. Thepresent study gives evidence that especially in cancer treatmentintra-tumoral injections will result in more efficient elimination ofthe tumor. The observation that monocyte-derived IL-3/IFN-β DC are ableto trigger apoptosis in tumor cells is relevant to their therapeutic useas anti-tumor vaccines. Indeed, recent reports demonstrated that humanIL4/GM-CSF DC can process apoptotic cells and cross-present the derivedantigens in a MHC-class I restricted fashion, resulting in the inductionof efficient cytotoxic T cell responses. Therefore DC which are directlyinjected into tumors will first induce apoptosis of cancer cells, andfinally migrate in the lymph nodes where they induce tumor-specificT-cell responses.

These compositions may, for example, be administered parenterally orintravenously. The compositions according to the invention forparenteral administration can be, in particular, sterile solutions,aqueous or non-aqueous, suspensions or emulsions. As a pharmaceuticallyacceptable solution or vehicle propylene glycol, polyethylene glycol,injectable organic esters, for example ethyl oleate, or cyclodextrinsmay be employed.

These compositions can also comprise wetting, emulsifying and/ordispersing agents.

The sterilisation may be carried out in several ways, for example, usingbacteriological filter, by incorporating sterilising agents in thecomposition or by irradiation. They may also be prepared in the form ofsterile solid compositions which may be dissolved at the time of use insterile water or any other sterile injectable medium.

The present invention can also comprise adjuvants which are well knownto a person skilled in the art (vitamin C, antioxidant agents, etc.)capable of being used in synergy with the compounds according to theinvention in order to improve and prolong the treatments of canceroustumors.

The invention also relates to a composition comprising a compositionaccording to present invention and another compound as a combinedpreparation for simultaneous, separate or sequential use for treatingcancer, infections and autoimmune diseases.

The present invention also relates to a method for the preparation of acomposition as described by present invention comprising followingsteps:

-   (a) isolating monocytes from a patient,-   (b) incubating said monocytes in a closed system in the presence of    clinical grade IFN-β and IL-3 to provide a population of IL3-R+    CD11c+ myeloid dendritc cells,-   (c) presenting an antigen on the surface of said dendritic cells in    clinical grade conditions, thereby providing a population of antigen    presenting dendritic cells; and,-   (d) activating a population of T cells with said population of    antigen presenting dendritic cells, whereby said IL3-R+ CD11c+    myeloid dendritic cells are unstimulated or stimulated using a    method according to present invention, or a combination thereof.

As described above each of these steps can be performed in vitro and/orin vivo.

Recently improvements were made for the production of DC inclinical-grade conditions. The present inventors described in Toungouzet al 1999⁴⁶ that the development of closed systems, avoidance ofexogenous proteins and respect of standard operating procedures (SOP) isneeded to be able to guarantee predefined specifications of the cellularproduct. In these documents a good manufacturing practice(GMP)-simplified procedure of IL4/GM-CSF DC generation fromleukapheresis products in a closed system, using synthetic culture mediadevoid of non-human protein is described. In analogy to this method,clinical grade IL-3/IFN-β DC can be prepared.

Unless other wise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention. All publications and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. The materials, methods, and examples are illustrative only andnot intend to be limiting. Other features and advantages of theinvention will be apparent from the following drawings, tables, detaileddescription, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIG. 1. IFN-β prevents IL-3Rα downregulation on cultured monocytes

Monocytes incubated in medium alone or with IFN-β (1000 U/ml) wereanalyzed by flow cytometry for the expression of IL-3Rα (CD123). Thickline: FACS profiles after staining with PE-conjugated anti-CD123antibodies. Dotted lines: FACS profiles after staining withisotype-matched control IgG1. Data from one representative experimentout of 3 on different blood donors.

FIG. 2. IFN-β cooperate with IL-3 to promote monocyte survival

Monocytes were cultured in medium alone (◯), or in the presence of 50U/mi IL-3 (∇), or 1000 U/ml IFN-β (●) or a combination of 1000 U/mlIFN-β and 50 U/ml IL-3 (▾). Apoptotic cells were enumerated by flowcytometry after staining for annexin V and propidium iodide (PI).Results were expressed as mean±SEM of percentages of cells negative forthe expression of both annexin V and PI.

FIG. 3. Ultrastructure of cells derived from monocytes cultured in thepresence of IFN-β and IL-3

Transmission electron microscopy of monocytes after culture for 6 dayswith either GM-CSF and IL4 or IL-3 and IFN-β (original magnification A ;×900, B ; ×1950). Both IL-3/IFN-β DC and GM-CSF/IL4 DC showed typicalappearance of dendritic cells including a lobulated nucleus, longcytoplasmic processes and tubulovesicular system. In the presence ofIL-3 and IFN-β, DC appeared as smaller cells filled with mitochondria.

FIG. 4. Expression of surface markers on cells derived from monocytescultured in IL-3 and IFN-β

(A) Phenotype of purified monocytes cultured for 6 days with eitherGM-CSF and IL-4 or IL-3 and IFN-β. Thick line: FACS profiles afterstaining with specific antibodies. Dotted lines: FACS profiles afterstaining with isotype-matched control antibodies. Data from onerepresentative experiment out of 6 on different blood donors.

(B) Phenotype of purified monocytes cultured with GM-CSF and IL-4, orIL-3 and IFN-β or IL-3 alone. Thick line: FACS profiles after stainingwith specific antibodies. Thin lines: FACS profiles after staining withisotype-matched control antibodies. Data from one representativeexperiment out of 6 on different blood donors.

FIG. 5. Expression of surface markers on cells derived from monocytescultured in IL-3 and IFN-α

Phenotype of monocytes cultured with either IL-3 and IFN-β or with IL-3and IFN-α. Thick lines: FACS profiles after staining with specificantibodies. Thin lines: FACS profiles after staining withisotype-matched control antibodies.

FIG. 6. Internalization of FITC-Dextran in IL-3/IFN-β DC

DC grown in GM-CSF and IL4 or in IL-3 and IFN-β were incubated in mediumcontaining 1 mg/ml FITC-Dextran for the indicated times and analyzed byflow cytometry. Results are from one representative experiment out of 3on different blood donors.

FIG. 7. DC-LAMP and CD83 are expressed on LPS-stimulated IL-3/IFN-β DC

Monocytes were cultured with either GM-CSF and IL-4 or IL-3 and IFN-β.DC were then stimulated or not with LPS (1 μg/ml) for 24 h. Thick lines:FACS profiles after staining with specific antibodies. Thin lines: FACSprofiles after staining with isotype-matched control antibodies. Datafrom one representative experiment out of 2 on different blood donors.

FIG. 8. T cell stimulatory activity of IL-3/IFN-β DC

(A) IL-3/IFN-β DC induce proliferation of naive CD4⁺ T cells. Cord bloodCD4⁺ T cells were cultured with allogenic IL-3/IFN-β DC (0) orGM-CSF/IL-4 DC (●) prepared from the same donors. After 5 days, T cellproliferation was quantified by [³H] thymidine incorporation. Data areshown as means±SEM of 6 independent experiments.

(B) Production of cytokines in mixed leucocyte cultures. Peripheralblood CD4⁺ T cells were cultured with allogenic IL-3/IFN-β DC (hatchedcolumns) or GM-CSF/IL-4 DC (open colums) at a DC:T ratio of 1:10. After6 days, culture supernatants were assayed by ELISA for determination ofcytokine levels. Data are shown as mean±SEM of 6 experiments.

*p<0.05 as compared to DC generated in GM-CSF and IL-4 (Wilcoxon'stest).

FIG. 9. Apoptosis of cancer cells using IL-3/IFN-β DC.

Human monocyte-derived DC generated from monocytes cultured in IL-3 andIFN-β were cocultured with [3H] thymidine-labeled tumor cell lines.After 18 h, intact nuclei were harvested and radioactivity was measured.Data are expressed as percentages of DNA fragmentation at 10:1 DC:targetcell ratio.

FIG. 10. ANTI-E7 response induced by IFN-β/IL-3 DC; as compared toGM-CSF/IL-4 DC

Anti E7 IFN-g response induced by 13 DC as compared to GM DC. Light barsrepresent response obtained with unstimulated DC whereas dark barsrepresent responses induced with stimulated LPS stimulated DC. ELISPOT,ELISA and cytometry were used as read-out systems. Results plotted arethose obtained after subtraction of the background (unloaded DC+autologous T cells).

TABLE 1. CYTOKINE PRODUCTION BY MONOCYTE-DERIVED DC

DC were generated in presence of either GM-CSF and IL-4 or IL-3 andIFN-β and activated with LPS (1 μg/ml) for 24h or with 3T6-CD40Ltransfectants for 3 days. Cytokine levels in supernatants were measuredby ELISA and results were expressed as means±SEM of 6 independentexperiments on different blood donors.

TABLE 2. Poly (I:C) Induces IFN-α Production by IL-3/IFN-β DC

DC were generated in presence of IL-3 and IFN-β, and activated eitherwith 3T6-CD40L transfectants for 3 days, or LPS (1 μg/ml) for 24h, orInfluenza virus for 24 h or Poly (I:C) for 48 h. IFN-α levels insupernatants were measured by ELISA and results were expressed asmeans±SEM of independent experiments on different blood donors.

EXAMPLES Example 1 Interleukin-3 and Interferon-β Cooperate to InduceDifferentiation of Monocytes into Dendritic Cells with Potent Helper TCell Stimulatory Properties

Materials and Methods

Cell Preparation and Culture

Peripheral blood mononuclear cells (PBMC) were obtained from heparinizedblood of normal donors by centrifugation over Lymphoprep densitygradient (Nycomed, Oslo, Norway). Monocytes were obtained either by 2hadhesion in 75 cm² flasks or by centrifugation over Nycoprep densitygradient (Nycomed), following by magnetic cell sorting using acommercially available monocyte isolation kit allowing to obtain cellsuspensions containing more than 95% monocytes (Miltenyi Biotec, Auburn,Calif.). To generate DC using IL-4 and GM-CSF (IL4/GM-CSF DC), purifiedmonocytes were cultured for 46 days in RPMI 1640 (Bio Whittaker Europe,Verviers, Belgium) supplemented with 2 mM L-glutamine (GIBCO, Paisley,Scotland), 20 μg/ml gentamicin, 50 μM 2-mercaptoethanol (GIBCO), 1%non-essential amino acids (GIBCO) and 10% fetal bovine serum (BioWhittaker Europe) in 75 cm² flasks or 6 well plates (7.5 10⁵ cells/ml)in the presence of GM-CSF (800 U/ml) and IL4 (500 U/ml) kindly providedby Schering-Plough (Kenilworth, N.J.), as described by Romani et al.⁴.In parallel, monocytes were cultured for 4-6 days in presence of IFN-β(1000 U/ml) (Ares Serono Europe, London, UK) and IL-3 (50 U/ml) (R&DSystems Europe, Oxon, UK), alone or in combination.

Electron Microscopy Study

Dendritic cells were fixed with 2% glutaraldehyde and Millonig'sphosphate buffer for 2 h. Cell blocks obtained after 1200 gcentrifugation for 10 min were postfixed with 2% osmium tetroxide andembedded in epoxy resin. Ultra-thin sections were stained withuranyl-acetate and lead citrate. Ultrastructural study was performedusing the electron microscope EMT400 (Philips, Eindhoven, Holland).

Flow Cytometry Analysis

For immunophenotyping, cells were washed in phosphate buffered saline(PBS) supplemented with 0.5% bovine serum albumin (BSA) and incubatedfor 15 min at 4° C. with one of the following fluoresceinated (FITC-) orphycoerythrin (PE)-conjugated monoclonal antibodies: FITC-anti-HLA-DRIgG2a, PE-anti-CD4 IgG1, FITC-anti-CD8 IgG1, PE-anti-CD11b IgG2a,PE-anti-CD11c IgG2b, PE-anti-CD14 IgG2b, PE-anti-CD33 IgG1,FITC-anti-CD45RA IgG1, PE-anti-CD45RO IgG2a, PE-anti-CD54 IgG2b,PE-anti-CD80 (B7-1) IgG1, PE-anti-CD123 (IL-3Rα) IgG1 and PE-anti-CD154(CD40L) IgG₁, all from Becton Dickinson (Mountain View, Calif.),PE-anti-CD86 (B7-2) IgG2b from Pharmingen (San Diego, Calif.),FITC-anti-CD3 IgG2a, FITC-anti-CD16 IgG1, FITC-anti-CD19 IgG1 andPE-anti-CD40 IgG1 from Biosource International (Camarillo, Calif.),FITC-anti-CD1a IgG2a from Dako (Glostrup, Denmark), andFITC-anti-HLA-Class I (A,B,C) clone B9.12.1 IgG2a, PE-anti-CD83 IgG2bfrom Immunotech (Marseille, France). Cells were also stained withcorresponding isotype-matched control monoclonal antibodies and thenanalyzed using a FACScan flow cytometer (Becton Dickinson).

Apoptosis Analysis

Apoptotic cell death was measured by flow cytometry usingFITC-conjugated annexin V (Becton Dikinson) and propidium iodide(Sigma-Aldrich, Bornem, Belgium) as per the manufacturer's protocol.

Endocytosis Assay

FITC-dextran (Molecular Probes, Eugen, Oreg.) was used to assess cellendocytosis as described by Sallusto et al.¹⁷. Briefly, cells wereincubated with 1 mg/ml FITC-dextran at 37° C. for 5, 15 or 30 min andthen analyzed using the FACScan flow cytometer.

Dendritic Cell Stimulation

DC (4×10⁵/ml) were stimulated by bacterial lipopolysaccharide (LPS) (1μg/ml), formaldehyde-inactivated influenza virus strain New Caledonia(kindly provided by N. Kuehm, Aventis, Pasteur Mérieux, Val de Reuil,France), or Polyinosinic-polycytidylic acid (Poly (I:C) (20 μg/ml)(Sigma), for 24 h or 48 h respectively, and culture supernatants werethen assayed for cytokine levels. In parallel, DC (2×10⁵/ml) wereactivated by co-culture with irradiated 3T6 fibroblasts transfected withthe human CD40L gene (CD40L transfectants) (5×10⁴/ml), and supernatantswere harvested after 3 days for determination of cytokine levels.

Mixed Leucocyte Cultures

DC were co-cultured in 96-well flat-bottom plates with allogenic naiveCD4⁺ T cells (2×10⁵/ml) isolated from newborn cord blood or adult PBMC.CD4⁺ T cells were purified by magnetic cell sorting using a commerciallyavailable CD4 T cell isolation kit (>95% purity as assessed by FACSanalysis) (Miltenyi Biotec). In the case of adult CD4+ T cells, a CD45RAisolation kit (Miltenyi Biotec) is further used for the enrichment innaive cells.

After 5 days, cell proliferation was assessed by [3H] thymidine uptakeduring the last 16h and culture supernatants were collected fordetermination of cytokine levels.

Determination of Cytokine Levels

ELISA kits were purchased from Biosource Europe (Fleurus, Belgium) fordetermination of TNFα, IL-6, IL-8 and IL-12 (p40) levels. Determinationof IL-12 (p70) levels was performed using a commercially available kit(Endogen, Woburn, Mass.). IFN-γ and IL-5 levels were measured bytwo-site sandwich ELISA using antibodies from Chromogenix (MöIndal,Sweden) and Pharmingen, respectively.

Statistical Analysis

Statistical Significance was Determined Using Two-tailed PairedWilcoxon's Test.

Results

IFN-β-treated monocytes maintain IL-3Rα expression and depend on IL-3for their survival As shown in FIG. 1 monocytes purified from PBMCexpress IL-3Rα (CD123) but lose this expression after 6 days of culturein medium alone. The loss of IL-3Rα expression was prevented when IFN-β(1000 IU/ml) was added on the first day of culture (FIG. 1). Thisanalysis was performed on the fraction of viable cells in the culture.Indeed, more than 90% of monocytes cultured in medium alone or inpresence of 1000 IU/ml IFN-β were apoptotic, as assessed by flowcytometry using double staining with annexin-V and propidium iodide(FIG. 2). The addition of IL-3 on the first day of culture dramaticallyenhanced monocyte survival. Indeed, more than 65% of cells were stillalive after 6 days of culture in presence of IL-3 and IFN-β.

Monocytes Cultured in Presence of IL-3 and IFN-β Differentiate into DC

To characterize the cells obtained by culture of monocytes in IL-3 andIFN-β, the inventors first looked at their ultrastructural morphology.As shown in FIG. 3, monocytes cultured under this condition acquiredcytoplasmic expansions of the dendritic type. Cells derived frommonocytes cultured in IL-3 and IFN-β will therefore be referredhereafter as IL-3/IFN-β DC.

Flow cytometry analysis (FIG. 4) demonstrated that these cells expressedmarkers of the myeloid lineage (CD11c, CD14, and CD33) but were negativefor the surface expression of CD3, CD4, CD8, CD11b, CD16, CD19, CD45RAand CD154 (CD40L). They also expressed high levels of HLA class I andclass II molecules, CD40, CD54, CD 80 and CD86, and IL-3Rα (CD123). Asexpected⁷, the latter marker was expressed at only low level on DCderived from monocytes cultured in IL-4 and GM-CSF. Conversely, CD1a wasexpressed on IL-4/GM-CSF DC but not on cells derived from monocytescultured in IL-3 and IFN-β.

When monocytes were cultured in IL-3 alone, they strongly adhered toplastic, so that only limited numbers of such cells could be collectedfor further analysis. By flow cytometry, they expressed lower levels ofCD80 and CD86 and higher levels of CD14 than IL-3/IFN-β DC (FIG. 4),which suggests that they are more close to macrophages. In additionalexperiments, the inventors found that IFN-α exerts a similar effect asIFN-β, as the IL-3+IFN-α combination also resulted in the generation ofmyeloid IL-3Rα-positive DC expressing CD80 and CD86 (FIG. 5).

The endocytosis capacity of IL-3/IFN-β DC was studied by fluid phaseuptake of FITC-dextran. As shown in FIG. 6, IL-3/IFN-β DC activelyengulf dextran, although they were less potent than IL4/GM-CSF DC indoing so.

To further study the maturation status of IL-3/IFN-β DC, the inventorsanalyzed their expression of CD83 and DC-LAMP which are establishedmarkers of mature DC. As shown in FIG. 6, both markers were absent onresting IL-3/IFN-β DC but were clearly upregulated upon LPS stimulation(FIG. 7).

Production of Cytokines by IL-3/IFN-β DC

IL-3/IFN-β DC spontaneously secreted IL-6, IL-8, IL-12 (p40) and TNFα.As compared to GM-CSF/IL-4 DC, IL-3/IFN-β DC produced less IL-12 (p40)whereas their secretion of IL-8 was slightly higher (table 1). As it isthe case for IL-4/GM-CSF DC, both LPS and CD40 ligation induced by CD40Ltransfectants upregulated the synthesis of cytokines by IL-3/IFNβ DC. Ascompared to GM-CSF/IL-4 DC, IL-3/IFN-β DC produced lower levels of TNF-αin response to LPS, higher levels of IL-6 and IL-8 in response to CD40L,and much lower levels of IL-12 (p40) and IL-12 (p70) regardless thestimulus considered.

To analyze the production of IFN-α, the inventors included as additionalstimuli formaldehyde-inactivated influenza virus and Poly (I:C) whichmimicks viral double-stranded RNA. As shown in table 2, Poly (I:C) wasthe only stimulus inducing IFN-α production by IL-3/IFN-β DC. Under thiscondition, IFN-α levels secreted strongly by IL-3/IFN-β DC were morethan ten-fold higher that those produced by IL-4/GM-CSF DC (128±24pg/ml, p<0.05) (FIG. 12).

T Cell Activation Induced by IL-3/IFN-β DC

To evaluate the ability of IL-3/IFN-β DC to elicit naive T cellresponses, mixed leucocyte cultures were prepared between cord bloodCD4⁺ T cells and either IL-4/GM-CSF or IL-3/IFN-β DC. At allstimulator/responder ratios, IL-3/IFN-β DC were as efficient asGM-CSF/IL-4 DC to induce naive CD4⁺ T cell proliferation (FIG. 8A). Insubsequent experiments designed to analyze the profile of cytokinessecreted by T cells upon exposure to allogenic DC, mixed lymphocytecultures were prepared using adult CD45RA⁺ CD4⁺ T cells as respondercells. As shown in FIG. 8B, IL-3/IFN-β DC induced the production oflarge amounts of IFN-γ much higher than to those elicited by IL-4/GM-CSFDC. To determine whether IL-12 was involved in the induction of IFN-γproduction, the inventors added a neutralizing anti-IL-12 antibody tothe mixed leucocyte cultures. IL-12 neutralization inhibits more than60% of the IFN-γ production whatever the DC type considered (data notshown), indicating that the low levels of IL-12 secreted by IL-3/IFN-βDC contribute to their ability to elicit IFN-γ production by T cells.Likewise, IL-3/IFN-β DC also induced IL-5 production in mixed leucocyteculture and were also much more efficient than IL4/GM-CSF DC in thatrespect (FIG. 8B).

Discussion

Among DC population, distinct lineages were defined according to theexpression of surface molecules. CD11c⁺ CD123⁻ DC display feature ofmyeloid lineage and depend on GM-CSF for their survival^(5;18), TheIL-3-dependent CD11c⁻ CD123⁺ DC are thought to belong to lymphoidlineage⁷ although Olweus et al. showed that in the T cell-dependentareas of human lymphoid organs, a large subset of DC expressing highlevels of IL-3Rα belong to a myeloid lineage¹⁹. The experimentsdescribed by the inventors demonstrate that monocytes cultured in IL-3and IFN-β give rise to a distinct type of DC expressing high level ofboth CD11c and CD123 surface molecules. In addition to their morphology,their dendritic cell nature was further established by their capacity toinduce proliferation of naive CD4⁺ T cells in MLR. Interestingly, IL-3was previously shown to cooperate with tumor necrosis factor in thegeneration of dendritic/Langerhans cells from CD34⁺ hematopoieticprogenitor cells²⁰. As the starting population in the experiments,performed by the inventors, consists of purified monocytes and theIL-3/IFN-β DC differ from the dendritic/Langerhans cells in terms ofCD1a and CD14 expression, it appears that IL-3 can promotedifferentiation of distinct DC populations.

Recently, IFN-β have been shown able to enhance maturation ofmonocyte-derived DC²¹. As compared to classical DC generated in GM-CSFand IL-4, IL-3/IFN-β DC are at a higher stage of maturation, asindicated by their increased surface expression of costimulatory and HLAmolecules. Decreased endocytic capacity of IL-3/IFN-β DC may alsoreflect their higher maturation level²². As far as cytokine productionis concerned, IL-3/IFN-β DC secrete much lower level of IL-12 ascompared to GM-CSF/IL-4 DC. This could be related to their higher stageof maturation, as DC maturation was previously shown to result in alower production of IL-12²³. Moreover, IFN-β might directly inhibitIL-12 synthesis by DC²⁴. The fact that IL-3/IFN-β DC exhibited lessendocytosis capacity may also reflect their higher maturation level.Although displaying several features suggestive of a higher degree ofmaturation than IL-4/GM-CSF DC, IL-3/IFN-β DC should not be consideredas fully mature as they do not express CD83 and DC-LAMP. However, thesemarkers clearly appeared upon LPS stimulation as in the case ofIL-4/GM-CSF DC.

A previous study observed that DC differentiated from monocytes culturedin presence of IFN-β and GM-CSF are short-lived¹⁶. In this report, thepresent inventors demonstrate that IL-3 rescue monocytes cultured inpresence of IFN-β from apoptosis, and allow them to differentiate intomature DC. These observations demonstrate a cooperating effect of IL-3and IFN-β on cell survival and differentiation. Little is known on theeffect of both type I and type II IFNs on IL-3 receptor expression. Theeffect of type I IFNs on IL-3Rα expression has never been assessed, butIFN-γ proved to upregulate IL-3Rα expression in human endothelialcells²⁵. Moreover, IFN-γ has been shown to have a synergistic effectwith IL-3 on the growth of immature human hematopoietic progenitors²⁶,though this effect was not correlated with upregulation of IL-3Rαexpression²⁷. Nevertheless, IL-3 is well known to stimulate monocytedifferentiation from myeloid progenitors and their activation²⁸⁻³⁰ .More recently, IL-3 proved to be a critical survival factor for IL-3Rαprecursors of DC isolated from human blood, lymph nodes and bonemarrow^(6;7;19).

Despite their low level of IL-12 production, IL-3/IFN-β DC stimulatehigh level of IFN-γ production from adult CD4⁺ T cells, suggesting thatthey use other factors or membrane molecules to elicit the synthesis ofTh1 cytokines. Indeed IL-12 independent pathway of IFN-γ were recentlydescribed^(13;14;31-33). However, the reduced IFN-γ levels measured uponIL-12 neutralization indicate that IL-12 contributes to the function ofIL-3/IFN-β DC. Taken together, the data as presented in presentinvention indicated that IL-3/IFN-β DC differ from the CD4⁺ CD3⁻ CD11c⁻IL-3Rα⁺ plasmacytoid cells isolated from peripheral blood described byGrouard et al.⁶, as the latter cells proved to be poor inducers of IFN-γin MLR.

The capacity of IL-3/IFN-β DC to produce high levels of IFN-α upon PolyI:C stimulation might be relevant to their effects in the setting ofviral infections³⁴. This response to Poly I:C is consistent with theirmyeloid origin³⁵. Interestingly, IL-3/IFN-β DC respond low to influenzavirus in relation with the induction of MxA by IFN-β³⁶. The differentialresponsiveness to influenza virus and Poly (I:C) is consistent with thefact that Poly (I:C) acts on the protein kinase R expressed in thecytoplasm whereas surface receptors are involved in responses to wholeviruses³⁷.

Monocyte-derived IL-4/GM-CSF DC are now used clinically as tools toinduce antitumor immunity³³⁸⁻⁴⁰. Herein, the inventors show thatIL-3/IFN-β DC are more efficient than IL-4/GM-CSF to elicit IFN-γ andIL-5 production by helper T cells. This might be relevant to cancervaccination as both cytokines were found to synergize in tumorrejection⁴¹. Because of their ability to induce the production of bothIFN-γ and IL-5 and their easy generation from peripheral bloodmonocytes, the inventors suggest that IFN-β/IL-3 DC might be of interestfor the development of new cancer therapies based on DC.

Summary of Example 1

The inventors observed that interferon-β (IFN-β) prevented thedown-regulation of the interleukin-3 receptor a chain (IL-3Rα) whichspontaneously occurs during culture of human monocytes. Thefunctionality of the IL-3R was demonstrated by the fact that IL-3rescued IFN-β-treated monocytes from apoptosis. Whereas more than 90%monocytes died after 6 days of culture in IFN-β alone, 65% of cells werestill alive in presence of IL-3 and IFN-β. The inventors then found thatmonocytes cultured in presence of IFN-β and IL-3 acquire a dendriticmorphology and express high levels of HLA class I and class II andcostimulatory molecules such as CD40, CD54, CD80 and CD86. Whenstimulated by either lipopolysaccharide (LPS) or fibroblasts expressingCD40 ligand (CD40L transfectants), DC generated in IFN-β and IL-3(IL-3/IFN-β DC) secreted high levels of IL-6, IL-8 and tumor necrosisfactor (TNF)-α, but only very low levels of IL-12 in comparison with DCgenerated in IL-4 and granulocyte-macrophage colony-stimulating factor(IL-4/GM-CSF DC). The inventors found that IL-3/IFN-β DC induced avigorous proliferative response of allogenic cord blood T cells. Inmixed leucocyte culture with adult CD4⁺ T cells, IL-3/IFN-β DC elicitedthe production of high levels of both IFN-γ levels and IL-5. Finally,IL-3/IFN-β DC were found to produce much higher levels of IFN-α thanIL-4/GM-CSF DC in response to Poly (I:C). The inventors conclude thatmonocytes cultured in presence of IL-3 and IFN-β differentiate into DCwith potent helper T cell stimulatory capacity despite their lowexpression of IL-12.

Example 2 Human Monocyte-derived Dendritic Cells Generated in IL-3 andIFN-β Induce Apoptosis of Tumor Cell Lines

It was proven by the inventors that human monocyte-derived DC generatedfrom periferal blood mononuclear cells by culture in interleukin (IL)3and interferon(IFN)-β (IL-3/IFN-β DC) may be used in clinical trials astool to induce anti-tumor responses in vivo⁻³⁸⁻⁴⁰. In order to determinewhether IL-3/IFN-β DC are able to induce programmed cell death in tumorcells, those cells were co-cultured with a panel of tumor cell lines(Jurkat cell line, the Molt-4 cell line (obtained from the InstitutPasteur, Lille, France); Cem cell line (obtained fron Dr. T. Velu (ULB,Brussels, Belgium), 786.0, A498 and Caki 2 cell lines provided by Dr. R.Kiss (ULB, Bussels, Belgium);Daudi cell lines purchased from ATCC). Thepercentage of DNA fragmentation into target cells was measured using thejam test. Briefly, target cells were labeled with 5 μCi/ml of [³H]thymidine by overnight incubation at 37° C. Labeled target cells wereharvested, washed and seeded in 96 well U-bottom plates at a density of10,000 cells/well. Effector cells were washed and added to the targetcells. After 18 h, intact nuclei (unfragmented high M.W. DNA) wereharvested using a micro 96 harvester and radioactivity was measured on amicro plate beta counter. Data were expressed as percentage of DNAfragmentation calculated by the following formula: [1-(cpm witheffector/cpm without effector)]×100. The present inventors observed thatIL-3/IFN-β DC at a ratio of E:T=10:1 exhibit significant cytotoxicactivity against 6 out 8 tested tumor lines. As shown in FIG. 9, Molt-4,Jurkat, 786.0, A498 and Caki2 cell lines were susceptible to IL-3/IFN-βDC-mediated apoptosis, as well as Daudi cells although to a lesserextent. On the other hand, Cem cells were resistant.

The observation that monocyte-derived IL-3/IFN-β DC are able to triggerapoptosis in tumor cells is relevant to their therapeutic use asanti-tumor vaccines. Indeed, recent reports demonstrated that humanIL-4/GM-CSF DC can process apoptotic cells and cross-present the derivedantigens in a MHC-class I restricted fashion, resulting in the inductionof efficient cytotoxic T cell responses⁻⁴²⁻⁴³. Therefore DC which aredirectly injected into tumors will first induce apoptosis of cancercells, and finally migrate in the lymph nodes where they inducetumor-specific T-cell responses.

Example 3 ANTI-E7 Response Induced by IFN-b/IL-3 DC as Compared toGM-CSF/IL-4 DC(FIG. 10)

Material and Methods

DC were generated from PBMCs of healthy blood donors by a 5 day culturein presence of either GM-CSF (800 IU/ml) and IL-4 (500 IU/ml) (GM DC orIL-4/CSF DC) or IL-3 (50 IU/ml) and IFN-β (1000 IU/ml) (13 DC orIL3/IFN-β DC). After collection, these cells were pulsed for 2 hourswith the E7 protein (20 μg/ml) at 370C in 5% CO2 atmosphere andactivated or not with LPS (1 μg/ml, overnight stimulation). E7 pulsed DCwere cocultured with autologous purified CD4+ T cells at a 1:10 ratiofor 6 days. ELISPOT, ELISA and flow cytometry (intra-cellular staining)assessing IFN-γ production were used as read-out system.

Results

The data of these experiments show that non-activated I3 DC are at leastequivalent or even superior to LPS stimulated GM DC for the induction ofanti-E7 responses. As E7 is a protein derived from HPV 16, a virusinvolved in the pathogenesis of the cancer of the cervix, these data arerelevant for the design of new cancer vaccines/immunotherapy.

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TABLE 1 Cytokine production by monocyte-derived DC Cytokine levels(ng/ml) Stimulation Cells TNFα IL-6 IL-8 IL-12 (p40) IL-12 (p70) NoneGM-CSF/IL-4 0.59 ± 0.26 0.31 ± 0.16 1.73 ± 0.75 <0.02 <0.01 IL-3/IFN-β0.81 ± 0.21 0.86 ± 0.17  6.40 ± 2.95* 0.29 ± 0.13 <0.01 LPS GM-CSF/IL-432.9 ± 7.13 16.6 ± 4.97 88.0 ± 14.3 68.7 ± 20.0 0.04 ± 0.01 IL-3/IFN-β 18.1 ± 3.22* 11.6 ± 2.59 69.6 ± 13.3  3.8 ± 1.53* <0.01* 3T6GM-CSF/IL-4 0.56 ± 0.55 0.45 ± 0.25 3.45 ± 1.27 0.12 ± 0.05 <0.01IL-3/IFN-β 1.08 ± 1.08 0.12 ± 0.10  12.0 ± 2.88* 0.03 ± 0.02 <0.013T6-40L GM-CSF/IL-4 2.86 ± 0.80 2.55 ± 0.45 15.0 ± 2.79 43.0 ± 19.0 0.38± 0.14 IL-3/IFN-β 4.06 ± 0.81  7.46 ± 2.17* 117.2 ± 41.1*  7.41 ± 1.50* 0.03 ± 0.01* *p <0.05 as compared to DC generated in GM-CSF and IL-4(Wilcoxon's test).

TABLE 2 Poly (I:C) induces IFN-α production by IL-3/IFN-β DC IFN-α levelStimulation n (pg/ml) None 11 <12 3T6-CD40L 11 <12 LPS 11 41 ± 9Influenza virus 6  90 ± 48 Poly (I:C) 11 1873 ± 236

1. A method for differentiation and maturation of isolated monocytesinto stimulated IL3-R+CD11c+ myeloid dendritic cells consisting of (a)incubating said monocytes with a combination of IFN-β and IL-3 and (b)incubating the IL3-R+CD11c+ myeloid dendritic cells with a stimulantchosen from a group consisting of virus, bacterium, lipopolysaccharide(LPS), nucleic acid, and a combination thereof.
 2. A method fordifferentiation and maturation of isolated monocytes into stimulatedIL3-R+CD11c+ myeloid dendritic cells consisting of (a) incubating saidmonocytes with a combination of IFN-β and IL-3 and (b) incubating theIL3-R+CD11c+ myeloid dendritic cells with poly (I:C).
 3. The methodaccording to claim 1, wherein IL-3 is present at a concentration between1 and 1000 U/ml.
 4. The method according to claim 3, wherein IL-3 ispresent at a concentration of 50 U/ml.
 5. The method according to claim2, wherein IL-3 is present at a concentration between 1 and 1000 U/ml.6. The method according to claim 5, wherein IL-3 is present at aconcentration of 50 U/ml.
 7. The method according to claim 1, whereinthe stimulant is a virus.
 8. The method according to claim 1, whereinthe stimulant is a bacterium.
 9. The method according to claim 1,wherein the stimulant is LPS.
 10. The method according to claim 1,wherein the stimulant is a nucleic acid.